MXPA97009740A - Process to recover insoluble compounds in water from a fermentac broth - Google Patents

Abstract

The present invention relates to a novel process for recovering water-insoluble compounds from a fermentation broth including the sequential steps of concentrating, solubilizing and diafiltering the compound of interest, all through a single closed recirculation system in order to recover the compound for additional current purification aba

Description

PROCESS FOR RECOVERING INSOLUBLE COMPOUNDS IN WATER FROM A FERMENTATION BROOD Field Technical The present invention relates to a novel process for recovering water-insoluble compounds from a fermentation broth. More specifically, this invention relates to a process for recovering cyclosporins from a fermentation broth. More specifically, this invention relates to a process for recovering cyclosporins and other valuable commercial products from a fermented broth. BACKGROUND OF THE INVENTION Several processes have been used in the past to isolate valuable water-insoluble commercial compounds from fermentation broths. Traditional technologies for isolating such compounds employ solid-liquid separations (eg, filtration, centrifugation, etc.) to isolate water-insoluble active ingredients and subsequent extractions of solids-liquids to recover activities. For example, The Patents of E. U.A. Us. 4, 1717, 1 18 and 4,215, 199 for Hérri describe the process for isolating cyclosporins A and B from fermentation broths involving the steps of centrifugation, homogenization and multiple extractions (using methanol, ethylene chloride and other water-immiscible organic solvents) with the corresponding evaporations (ie, concentrations). Therefore, final extracts are subjected to chromatographic purifications using silica gel and packages of SEPHADEX® LH20. Similar procedures are employed to isolate other types of water-insoluble compounds such as lovastatin (an anti-hypercholesterolemic) and tacrolimus (FK-506, an immunosuppressant). Although these methodologies are currently used for industrial scale fermentations, they usually require solid-liquid separators and extractors / evaporators / solvent condensers that have high energy requirements. In addition, product recovery returns from these processes are low due to multi-stage operations. Therefore, the capital investment and subsequent production costs are high. As another example, the Rudat and gold patent (U.S. Patent No. 5,256,547) describes a process for the production and isolation of cyclosporin A which involves mixing the culture with a filter aid such as modified gypsum or calcite flour to form a suspension and filter the mixture to obtain a wet biomass. The biomass is then dried and extracted two or three times with a lower carboxylic acid ester, or alternatively with a supercritical gas such as carbon dioxide. The extract is then defatted and chromatographed by preparative HPLC using silica gel or alumina oxide. This method offers limited advantages over those described in the prior patents as it still suffers from multiple, complicated and costly operations. Although the use of either microfiltration (MF) or ultrafiltration (UF) to classify / filter aqueous fermentation broths has been established in the literature, extractions with organic solvents are usually carried out as a secondary purification step to recover the active product As noted above, conventional purification procedures involve two different unit operations, namely separation and extraction / evaporation. Generally for water insoluble products, the compound is first isolated from the large volume of aqueous fermentation broth and then purified by repeatedly removing the compound with solvent and evaporating the solvent, so that the compound can be further extracted with a different solvent and evaporated to that a concentration is achieved from which the final purification takes place. Repeated extractions and evaporations, however, make the process for large-scale manufacturing very costly. A single aspect of the present invention is to have a continuous processing system that obviates the need to separate the extraction and evaporation steps after the initial centrifugation and / or filtration step. This technology offers many advantages over the processes of the prior art, including simplicity of design, reduced capital and manufacturing costs and increased recovery performance. In addition, unlike the traditional processes, the entire process of the present invention is automatic and is completely contained which reduces exposure to both people and the environment to the compound. This is an important consideration in that immunosuppressants and other potent therapeutic compounds can be highly toxic.

SUMMARY OF THE INVENTION It is therefore an objective of this invention to provide a process for the recovery of water-insoluble compounds derived from fermentation broths. It is another object of this invention to provide a process for recovering cyclosporins and other drugs from a fermentation broth containing them. It is another object of this invention to provide a less expensive process for the large-scale recovery of cyclosporins and other drugs from a fermentation broth. Other objects of this invention will be apparent to Iso skilled in the art of the present disclosure. In summary, the invention relates to a process for recovering a water-insoluble compound from a crude fermentation broth, comprising the steps of: a. concentrating the fermentation broth by tangential filtration through a compatible porous filtration membrane of solvents, to produce a permeate that traverses the membrane and a retentate comprising the concentrated broth, the water-insoluble compound being retained in the retentate wherein the retentate recirculates continuously along the circulation path to form a retentate stream, where the raw broth is fed to the retentate stream until all the raw broth is concentrated; b. solubilizing the insoluble compound in retentate water by adding a solvent to the concentrated broth to produce a solution of the compound; and c. filtering or diafiltering the solution through the porous membrane of step (a) to produce a permeate of solvent passing through the porous membrane wherein the solvent permeate comprises the solubilized compound. Optionally, the solvent permeate can be further concentrated using an osmosis or reverse ultrafiltration membrane and purified by any method known to those skilled in the art. BRIEF DESCRIPTION OF THE INVENTION Figure 1 shows a diagrammatic representation of a modality of the invention. Detailed Description of the Invention The process of the present invention is directed to the recovery of water-insoluble compounds that are produced by large scale fermentations. "Recovery" as used herein, refers to the process for removing materials without compounds from the compound of interest and encompasses the removal of excess fluid (e.g., concentration by elimination of fermentation broth) and / or removing dissolved or insoluble impurities.

Although the removal of fluid and impurities from the compound of interest results in some purification of the compound, it should be noted that "recovery" does not require achieving any particular degree of purification. That is, recovery does not necessarily result in the compound that complies with a defined purification standard (such as a National Formulary specification, United States Pharmacopeia or European Pharmacopeia); rather, the removal of fluid and impurities by itself is sufficient to achieve recovery. A condition for the use of the invention is that the compound itself is insoluble in the fermentation broth at the end of the fermentation. "Insoluble compound", as used herein, refers to either a solid compound dispersed in a liquid or gas or any emulsion of said compound. The insolubility of the compound can result either from the natural properties of the same compound, or as a consequence of adjusting the pH of the solution or ionic conditions. For example, immunosuppressants such as cyclosporins are normally produced as insoluble products under fermentation conditions. However, certain antibiotic compounds such as erythromycin are usually soluble in the growth medium used to grow the production organism, but can become insoluble at the end of the fermentation process by increasing the pH of the broth to about 8.7-11.0. However, whether the insolubility of the compound is inherent to the compound itself or results from the particular solution conditions, someone with ordinary skill in the art could easily understand that the process of the present invention is applicable to any compound insoluble in water, existing in suspension or as an emulsion. Examples of water-insoluble compounds include, but are not limited to, antibiotics (such as erythromycins A, B, C, and D), immunosuppressants (such as cyclosporins A, B, and G, rapamycin, ascomycin, or tacrolimus), anti-hypercholesterolemic growth hormones. (such as lovastatin, pravastatin or simavastatin) and any intermediates and / or derivatives thereof. The manner in which the fermentation is carried out is not important for the invention, in that any known condition of fermentation can be used. In most circumstances, and particularly for large-scale industrial fermentation, the culture medium and fermentation conditions (organism strain, inoculum type, fermentation time, fermentation temperature, etc.) are optimized to produce a yield maximum of the desired compound. Examples of suitable fermentation parameters for the production of cyclosporin A and B are described in U.S. Patent Nos. 4,117,118 and 4,215, 199 to Hárri and others, and 5,256, 547 to Rudat et al.; Fermentation parameters suitable for the production of lovastatin, simvastatin, pravastatin of antihypercholesterolemic and the like are described in the Patents of E.U.A. Nos. 4,231, 938 of Monaghan and others, 4,444, 784 for Hoffman and others and 4, 346,227 of Terahara and others; any suitable fermentation parameters for the production of the immunosuppressant tracolimus (FK-506) are described in the patent of E. U.A. No. 4, 894, 366 of Okuhara and others, all of which are incorporated herein by reference. For the purposes of the present invention, the fermentation process itself can be carried out in any small and large scale fermentation apparatus, varying in size from 10 liters to 100,000 liters. At the end of the fermentation, the fermentation broth containing the desired compound is placed in contact with a solvent-compatible filtration membrane and filtered by tangential filtration. "Tangential filtration" as used herein, refers to the process for passing a suspension (such as a fermentation broth) through a porous filtration surface in a substantially continuous flow and under pressure so that a large portion of the liquid passes through the filtration membrane. The suspension portion that passes through the filtration membrane is referred to as "permeate" or "filtrate"; that portion of suspension that does not pass through the membrane is named "retentate" or "concentrate." The water-insoluble compound of interest remains in the retentate. It should be noted that the filtration process does not require the complete removal of all the aqueous medium from the insoluble compound, i.e. the retentate may also comprise some residual fermentation broth. However, the permanent aqueous medium may decrease the solubilization efficiency of the solvent in the subsequent step (described below) due to a dilution effect. This, in turn, may require the use of more solvent to achieve the same degree of concentration efficiency. The filtration membrane can be made of any material capable of supporting (ie, not deteriorating under) the particular solution conditions existing at the end of the fermentation process, ie, high or low acidity, high or low alkalinity, high or low temperature, high pressure and the like. In addition, when the same filtration membrane is used in the subsequent filtration step (see below), it must be "solvent compatible", ie, the filtration membrane must resist degradation when in contact with the particular solvent for used in order to solubilize the compound of interest (as discussed below). Any commercially available filtration membrane can be used for tangential filtration, although surface or non-deep type membranes are preferred. "Surface-type" or "non-deep" membranes are those membranes that retain particles on their surfaces instead of absorbing or capturing particles on or within the structural matrix of the membrane. Suitable filtration membranes include polymeric structures compatible with organic solvents made of cellulose, polystyrene, polysulfone or polyamide. Preferred microfiltration membranes are DURAPORE® HVPP membranes compatible with organic solvents (manufactured by Milipore Corporation, Bedford, MA 01730) or ceramic structure composed of alumina. A more preferred microfiltration membrane is ceramic alumina. Ceramic alumina filters such as MEMBRALOX® can be purchased in U.S. Filter Corporation, (181 Thorn Hill Rd., Warrendale, PA 15086-7527). The ultrafiltration membranes compatible with suitable solvents include PZHK membranes (200,000 molecular weight classification) also available from Millipore Corporation. The pore size of the first filtration membrane is selected according to the particle size of the desired insoluble compound contained in the fermentation broth at the end of the fermentation process. Due to their hydrophobic nature, the water-insoluble compounds self-aggregate to form particles in aqueous solution, or form aggregated structures in association with the structures of their respective production organism (eg, cell wall components, mycelia, etc.). ). Therefore, the pore size of the membrane in the present invention is selected to retain desired insoluble particulate materials and to allow other smaller sized matter (when present) as well as soluble compounds to pass through as the permeate aqueous. "Particulate material" as used herein, refers to the desired insoluble composition in a self-aggregated form, or to the desired compound physically and / or chemically associated with any undesired insoluble matter or particulate. For example, cyclosporins are physically associated with mycelia at the end of the fermentation process. A similar phenomenon occurs with the immunosuppressant tracolumius (FK-506). Therefore, for these compounds, the pore size is selected in order to retain the particulate mycelium / compound materials instead of the specific compound itself. Filtration membranes of variable pore sizes can be employed in the first filtration step depending on the size of the particulate matter of interest. Preferred microfiltration membranes (particularly for retaining cyclosporin mycelia) have pore sizes ranging from about 0.02 to 5.0 μm, while ultrafiltration membranes have pore sizes ranging from about 0.001 to about 0.05 μm. However, it is understood that one skilled in the art can easily select a suitable membrane for any desired particulate matter of known size. In addition, in the interest of efficiency, it is generally convenient to use the larger pore size that still retains insoluble particulate materials (since the larger the pore size, and the faster the flow rate, the others are the same). terms). Therefore, in addition to the micro and ultrafiltration membranes, larger pore filtration membranes are contemplated within the invention, as long as they retain the compound of interest and are suitable for tangential filtration. Optionally, other filtration conditions can be optimized (once the filtration membrane has been selected to increase the processing efficiency of the compound and to minimize processing costs, for example, having selected a membrane with a particular pore size. , other filtration variables such as transmembrane pressure, cross flow regime and temperature will be empirically correlated with a permeate flow regime (permeate flow regime, also known as permeation regime, refers to the volume of permeate generated by filtration on A given surface area of membrane and over a given time, this regime is usually expressed in units of liters / square meters / hour (L / m2 / hr). By adjusting the filtration variables, the permeate flow rate can be optimized to reduce the amount of membrane required, for example, a fermentation broth can have a régime n non-optimized permeate flow of 10 l / m2 / hr. Therefore the filtration of 1000 liters of broth in a period of 10 hours (100 l / hr), could require 10 m2 of membrane (given that 100 l / 10m2 / hr = 10 L / m2 / hr). However, optimizing the permeate flow rate at 100 L / m2 / hr, only 1 m2 of membrane could be required to achieve the same result (ie, the filtration of 100 liters of broth in a period of 10 hours). Since the cost of the membrane itself can contribute significantly to the overall cost of large volumes of broth processing, the reduction of the surface area of the membrane is a particularly important consideration for increased operations. After the initial filtration, in retentate it can optionally be diafiltered with approximately two to four volumes of water (relative to retentate) to further remove water-soluble impurities. The "diafiltration" or "diafiltration" as used herein, refers to a special case of tangential filtration, that is, to the process of adding a liquid to the retentate at a rate approximately equal to the permeation regime so that the Retentate is maintained at a generally constant volume during tangential filtration. "Diafiltration" is a term analogous to permeate, and refers to that portion of suspension that passes through the membrane during the diafiltration process. During diafiltration, the residual fermentation broth in the retentate is continuously diluted so that the diafiltration further purifies the desired insoluble compound from the residual soluble contaminants in the broth. Furthermore, depending on the content of solids, both the degree of concentration and the diafiltration volume can be varied to minimize the possibility of the membrane becoming stuck, to reduce the time of the process and to maximize the passage of the product. In the second step of the inventive process, the retentate is mixed with a solvent capable of solubilizing the compound of interest to form a solvent slurry. The solvent and its volume are selected so as to preferentially solubilize the compound of interest and minimize the solubilization of other insoluble compounds as well as minimize the extraction of any soluble impurities present in the retentate. Solvents useful in the present invention include alcohols, lower ethers, lower ethers, lower ketones and certain chlorinated hydrocarbons such as chloroform and methyl chloride. Preferred solvents include lower alcohols, esters, ether and ketones wherein "lower" refers to straight or branched chain hydrocarbons of 1-6 carbons. Examples of lower alcohols are methanol, ethanol, butanol propanol and pentanoi; examples of lower esters are methyl acetate, ethyl acetate and methyl butanoate; examples of lower ethers are methyl ethyl ether, diethyl ether and 2-methoxypentane and examples of lower ketones are propanone, 2-butanone and 3-pentanone. Preferred solvents for cyclosporins include primary or lower secondary alcohols and propanone. Those skilled in the art can easily select a suitable solvent knowing the chemical and physical properties of the compound of interest. The amount of solvent used generally is at least equivalent to the amount of retentate that remains at the end of the first filtration, but can largely exceed it. Normally, two to six equivalent volumes are used. The efficiency of solubilization depends on the volume of solvent, that is, the more solvent is used, the greater the product will recover from the retentate. However, it is also preferred to use as little volume of solvent as possible to minimize the volume of permeate that may be needed to concentrate in an additional downstream step (as discussed below). The solvent is mixed with the retentate for a sufficient time to solubilize the majority of the water insoluble compound of interest. Although this time can vary from 0 to 24 hours, normal periods vary from approximately two hours to around six hours. However, it is understood that the optimum mixing time may vary, depending on the amount of retentate present, the compound of interest, its solubility and the solvent and volume of solvent used. In the third step of the process, the solvent slurry is filtered by tangential filtration through a porous filter membrane compatible with solvent. The filtration membrane is preferably the same filtration membrane as used in the first step of the process but a fresh or different filtration membrane can also be used (its compatible solvent is provided). Since the desired compound is now dissolved in solvent, the solvent permeate, instead of the retentate, is collected during the filtration process. Although the grout is discarded at the end, after the permeation, the grout can optionally be diafiltered with additional solvent. In a manner similar to the aqueous diafiltration described above, the diafiltration of solvents achieves by adding solvent to the residual slurry at a rate approximately equal to the permeation rate. In this situation, the additional solvent serves to further extract the residual non-solubilized compound that remains in the slurry. The subsequent solvent diafiltrate obtained is combined with the solvent permeate and constitutes a combined solvent permeate. The additional concentration of the combined solvent permeate can be achieved optionally by filtering it tangentially through a compatible solvent membrane having a different pore size than previously used. The retention membrane used in this step is selected to retain the desired compound based on the solubilized size of the compound (ie, molecular weight) instead of its particle size (insoluble aggregate) and to allow the solvent to pass through. the membrane as permeate (which is discarded). The ultrafiltration (UF), nanofiltracon (NF) or reverse osmosis (Ol) membranes having specific molecular weight reductions (RPME) are used for this purpose. U F / RPM E membranes suitable for concentrating the collected permeate include regenerated cellulose acetate membranes in a frame and plate or spiral type configuration, which are commercially available from Millipore Corporation. The NF or OI / RPME membranes include MPS SeI RO ™ series cartridges developed by Membrane Products Kyryat Eizmann Ltd. (P.O. B. 138, 76101 Rehovot, Israel) and are distributed in E.U.A. by LCI Corporation (P.O. Box 16348 Charlotte, NC 28297) and also include spiral wound cartridges of NANOMAX ™ series cellulose acetate also available from Mi llipore Corporation. After concentration in the membrane, the product can optionally be further processed by crystallization or chromatography, in the case of purification by chromatography, the solution can be contacted with a chromatographic medium that selectively retains the compound of interest contained in the solution . Typically, said chromatographic medium is a microporous matrix (prepared from copolymerization of styrene and divinylbenzene) or a porous silica gel or alumina oxide. The matrix should be a surface area large enough to join the desired components of the product feed. The chromatography medium useful in the present invention includes packages, supports or polymeric resins. Examples of such chromatographic media include SE PHAROSE®, SEPHADEX® and SEPHACRYL®, (available from Pharmacia Biotech Incorporated, 800 Centennial Ave., PO Box 1327, Piscataway, NJ 08855-1327), DOWEX series medium (available from Dow Chemicals, Midland, Ml), BIO-REX®, MACROPREP® and BIO-GEL® series media (available from BioRad Laboratories, 85A Marcus Drive, Melville, NY 11747) and Tentacle series packages (available from EM Separations Technology, 350 Columbia St ., PO Box 352, Wakefield, RL 02880). An example of a non-functional polymeric package is AMBERCHROM ™ CG161-m which can be purchased from TosoHass (Independence Mail West, Philadelphycia, PA 19105). As another optional step, the concentrated compound can be purified a second time with a suitable chromatography medium such as silica gel or reverse phase C8 or C18 packages. Suitable chromatography means for such purifications are well known to those skilled in the art. In an optional final step of the process, the compound can be extracted in another organic solvent, concentrated and crystallized. The crystals are then separated by filtration or centrifugation and dried under vacuum to obtain the final purified product. In a preferred embodiment and particularly for large-scale fermentations, the isolation of the desired water insoluble compound is achieved in a closed circulation system as shown in Figure 1. The fermentation broth (A) is introduced via a first port of input (1) in the system comprising a receiving bank (2) a first connection pipe (4) extending from the output port (3) of the receiving tank (2) to the input port (6) of the module filtration (7), a pump (5) to pump the fermentation tracing (cure or concentrate) through the first connection pipe (4), a filtration module (7), a filtration membrane (8) housed inside of the filtration module (7) and a second pipe (10) extending from the outlet port (9) of the filtration module (7) to a second input port (11) of the receiving tank (2). In operation, the fermentation broth (A) enters the receiving tank (2) first through an inlet port (1), where it flows, under pressure created by the pump (5), into and through the first connection pipe (4) to the filtration module (7). Inside the filtration module (7), the broth is brought into contact with the filtration membrane (8). the broth passes through the filtration membrane (8) and is filtered by tangential filtration to produce a permeate (B) which is discharged through an outlet port (12) and a retentate (C). The retentate (C) then enters a second connection pipe (10) extending from the outlet port (9) of the filtration module (7) to the second input port (11) of the receiving tank (2). The concentrated broth (C) enters the receiving tank (2) where it is mixed with non-concentrated fermentation broth that enters (A). Therefore, the circulating broth forms a retentate stream, flowing unidirectionally through the closed system. In the preferred mode, the system is designed to circulate the fermentation broth under a transmembrane pressure (PTM) of approximately 0.219-3.5 kg / cm2 and at a controlled temperature of approximately 30-60 ° C. The broth is circulated through the closed system until approximately one-quarter to one-half the volume of the starting broth remains as a retentate. In order to minimize the problem of the membrane becoming clogged (resulting from the concentration of the broth), the system can optionally be designed to incorporate a reinforcing pulsation mechanism that serves to periodically force the permeate boosters through the membrane. filtering element. As a result of subsequent pulsation, the shock layer is lifted off the membrane and transported away in the transverse flow of the retentate. As an alternative or in addition to a subsequent pulsation mechanism, the system design can incorporate any fed and bleeding configuration known in the art. These configurations help to avoid the local overconcentration of the retentate in the membrane. Therefore, somebody skilled in the art can easily adapt the system to prevent the membrane from sticking by any known method. It should also be noted that the described operation system can be configured and / or significantly lengthened to accommodate large volumes of broth and minimize processing costs. For example, the operation system can be designed with several filtration modules (in parallel or in series), multiple pumps, ducts and receiver tanks. Large operating systems can be partially or fully automatic. In addition, large operating systems may incorporate additional downstream purification step as part of a global recovery / purification scheme. Therefore individuals who have ordinary experience in the chemical engineering arts could easily increase or adapt the operating system to conform it with available resources (ie, equipment and space) and to contain manufacturing costs. In the second step of the process, the permeate outlet port (12) of the membrane filtration module (7) is closed. Then, a suitable solvent is added to the retentate in the receiver tank (2) through the same inlet port (1) as the raw fermentation broth. The solvent is mixed with the compound of interest for two to six hours until the majority of the compound dissolves. In the next step, the outlet port is reopened and the solvent slurry is recirculated through the closed system where it is contacted and filtered through the filtration membrane of the first step. Unlike the first step, where the aqueous permeate contains little of the compound, the permeate of the solvent contains the majority of the compound as a dissolved product. Therefore, it is collected continuously in a storage tank to be processed further downstream. In the preferred embodiment, once the retentate stream is re-started, fresh solvent is added continuously in the receiving tank to maintain a constant volume of slurry. In other words, the solvent slurry is diafiltered with fresh solvent to continuously extract any residual product in the liquid phase. The diafiltrate of the solvent is then combined with the permeate of the solvent in the separate storage tank. The end of the solvent diafiltration step, the addition of dry solvent is stopped and the solvent slurry is further concentrated by filtration alone. This step allows a manufacturer to recover the maximum amount of product from the slurry before discarding the spent slurry as waste. . If necessary, a fourth step can be designed in such a way that water can be added to spent leftover concentrate to recover the residual solvent which may not be suitable for anaerobic waste treatment (since the presence of organic solvent usually increases the DOB (biological or biochemical oxygen demand)., the solvent recovered from this washing step can be recycled or distilled to be reused in order to minimize the impact to the environment. This then completes the three cycles of membrane operations summarized in the following Table 1. The combined solvent permeate (ie, solvent permeate plus solvent diafiltrate) is subsequently concentrated using an NANOMAX series spiral wound coil cartridge. Millipore ™. After which the concentrate can be further purified by recrystallization or chromatography in a normal example, a column of AMBERCHROM ™ CG161-m is loaded with a quantity of crude cyclosporin concentrate and the bed is eluted with an ethanol-gradient. water (20-60%). The individual fractions are analyzed by high performance liquid chromatography (HPLC) or thin layer chromatography (TLC) to determine the fractions containing compound activity. The chromatography solution and / or all the combined fractions of eluted therefrom, can then be concentrated either by ultrafiltration or reverse osmosis and further purified by any method known to those skilled in the art. The concentrated solution can also be extracted with a suitable solvent to prepare the final purification. Table 1 Cycle No. Process Food Retentate Permeate 1 Concentration of Broth Broth (product) Aqueous waste Compound Fermentation 1a * Diafiltration Water Broth (product) Aqueous waste 2 Solubilization / Solvent Grout (product) None Mixed 3 Filtration None Grout (product Clarified residual liquid) (product) 3a "Diafiltration Solvent Slurry (product Clarified Liquid Fresh residual) (product) 3b * Concentration of None Grout (product Clarified residual grout liquid) (Product) 4 * Washed Water Water Washed Solvent Reclaimed * Refers to optional steps The invention will now be further described as examples. The examples are merely illustrative of the invention and are not intended to limit the invention in any way. Example 1 Recovery of Cyclosporins by Ceramic Microfiltration and Extraction Method Approximately 160 liters of cyclosporin fermentation broth from Operation CD-263 containing 5.1% dry solids and 10% wet suspended solids was fed into a receiving tank. The membrane unit consisted of two μ ROMICON® (Koch Membrane Systems, Inc., 850 Main Street, Wilmington, MA 01887) ceramic microfiltration membrane (MFC) membrane elements (in series), each having a surface area of 0.2 m2. The inlet pressure was set at 4.21 kg / cm2 and the broth was recirculated through the system while the aqueous permeate containing the water soluble impurities was removed. After 90 minutes, during which time the average flow of permeate was 183 L / m2 / hr, the volume was concentrated to approximately 50 liters., Then water was added to the receiving tank at the same rate as the permeation rate for Continue the removal of water and associated impurities. The permeate flow rate was measured at approximately 150 L / m2 / hr. Approximately 155 liters of permeate and diafiltrate were collected and sampled by HPLC; little cyclosporin activity was detected in the sample. As a second step, 100 liters of methanol were added to the receiving tank which contained 50 liters of concentrated broth (diafiltrate), the valve for the membrane was closed and the slurry was mixed for two hours. The MFC unit was restarted and the dissolved product was separated by the membrane (i.e. as a permeate) and collected in a product tank. The permeate flow rate started at 105 L / m2 / hr under pressure of 4,218 kg / cm2 (inlet) and 2.24 kg / cm2 (outlet) and slowly decreased to 36 L / m / hr. The pressure was then increased to approximately 5.64 kg / cm2 (inlet) and 3.86 kg / cm2 (outlet) while the temperature was maintained at 28-30 ° C. After concentrating the slurry to 100 liters, 20 liters of methanol wash were added (in order to increase the yield of cyclosporin). Approximately 27 grams (64%) of cyclosporin was recovered while 15 grams (36%) were still present in the retentate after the methane wash, as determined by CLAR. Example 2 Recovery of Cyclosporins by Ceramic Microfiltration and Methanol Extraction / Ethyl Acetate Approximately 8 liters of cyclosporin fermentation broth from Operation CD-265 containing 4.6% dry solids was fed to a receiving tank. The membrane unit consisted of a CERAFLO® ceramic microfiltration membrane (MFC) element having a surface area of 0.12 m2. The outlet pressure was set at 1.4-2.1 kg / cm2 and the inlet pressure at 3.51-3.8 kg / cm2. The broth was recirculated through the system while stirring the aqueous permeate containing the water soluble impurities. The rate of initial permeate flow through the membrane was 450 L / m2 / hr and slowly decreased to 100 l / m2 / hr. Then, a total of 12 liters of distilled water were added in aliquots of 2 liters to the tank at the same rate as the permeation regime (diafiltration) to continue the removal of water and associated impurities. The flow rate was measured at approximately 50-65 l / m2 / hr. As a second step, the valve for the membrane was closed and 2 liters of methanol were added to the receiving tank; the slurry was then mixed for two hours. Two additional liters of methanol / ethyl acetate (50/50 v / v) were added before the CMF unit was restarted. The cyclosporin product dissolved in the methanol / ethyl acetate solvent was diafiltered through the membrane and collected in a product tank. The flow rate was started at 75 L / m2 / hr under pressure of 3.37 kg / cm2 (inlet) and 1.82 kg / cm2 (outlet) and slowly increased to 155 L / m2 / hr through diafiltration with a total of 16 liters of methanol / ethyl acetate The temperature was not controlled and fluctuated between 28-36 ° C during the diafiltration of the solvent. EXAMPLE 3 Methanol Extraction of Cyclosporin A using 0.05 μm of Ceramic Memories N i ro® Approximately 140 liters of cyclosporin fermentation broth from Operation CD-268 containing 1 1 .3% dry solids and 26% Suspended wet solids were fed to a receiving tank. The membrane unit consisted of a 0.05μ element of ceramic microfiltration membrane (M FC) N i ro® having a surface area of 0.3 m2, and channels of 6 mm (mm) in diameter. The inlet pressure was set at 4.38 kg / cm2 and the broth was recirculated through the system while stirring the aqueous permeate containing the water soluble impurities. The initial permeate flux rate through the fe membrane of 2.46 l / m2 / hr and decreased slowly to 48 l / m / hr after 90 minutes. The volume of the broth was concentrated to approximately 58 liters, then 72 liters of distilled water were added to the receiver tank to continue the removal of water and associated impurities. The permeate flow rate increased approximately to 220-280 l / m2 / hr due to the dilution effect. After concentrating the diluted broth to 40 liters, an additional 90 liters of distilled water was added to continue the washing of the broth. The collected permeate was analyzed by HPLC and showed little activity (approximately 0.002 grams / L cyclosporin A). When the broth (retentate) was concentrated to 38 liters, the ceramic unit was stopped and 92 liters of methanol were added and mixed for about 14 hours to dissolve the cyclosporin in the alcohol phase. The MFC unit was restarted and the product dissolved in the methanol permeate was separated by the membrane and collected in a product tank. The flow rate of the solvent permeate started at 50 L / m2 / hr under pressure of 4.7-4.9 kg / cm2 (inlet) and 2.6-2.8 kg / cm2 (outlet) and slowly decreased to 14 l / m2 / hr. The temperature was started at 35 ° C and slowly increased to approximately 50 ° C from the heat generated by recirculation. A total of 70 liters of methanol containing the product was collected for further processing. Example 4 Ethanol Extraction of Cyclosporin A Using a Niro® 0.05μm Ceramic Membrane Approximately 30,500 L of fermentation broth from operation 102 was pumped into a receiving tank which fed (four recirculation cycles containing each four) into Niro® ceramic microfiltration (MFC) series modules (housing 0.05 μm membranes with a total surface area of 60 m2 for the system). The transmembrane pressures were controlled at approximately 0.35-1.05 kg / cm2 and the feed temperatures were controlled from 35 ° C to 45 ° C. The permeate flow rates varied from 23 L / m2 / hr to 62 L / m2 / hr. The material was concentrated at 9,000 I and with approximately 24,000 L of water. The permeate flow varies from the scale of 44 L / m2 / hr to 70 L / m2 / hr. The material was concentrated to a final volume of 8,100 I.

Approximately 10,900 I of Especially Denatured grade 3A ethyl alcohol was then added to the concentrate and heated at 35 ° C to 40 ° C for two hours. The solvent slurry was diafiltered with 19,000 additional grade 3A Especially Denatured ethyl alcohol. The slurry was concentrated to a final volume of about 6,500 I and contained less than 5% of the raw broth activity determined by HPLC. Approximately 35,000 I of solvent permeate was collected and subsequently concentrated by reverse osmosis described in the following Example 5. Example 5 Concentration of Cyclosporin A using a Millipore NANOMAX ™ -50 Reverse Osmosis Membrane. Approximately 35,000 I of ethanol solution enriched with cyclosporin from Operation 102 was fed to a Millipore reverse osmosis unit containing total surface area of membranes 180 m2 of NANOMAX ™ -50. The membranes are compatible with up to 70% ethanol (by weight), therefore the fed stream was partially diluted with water and sorted by filtration before feeding unit reverse osmosis. The material was pumped from a feed tank to a multi-stage high pressure pump. The product was pumped through the membranes at cross flow rates of 120-170 L / min and the retentate was returned to the feed tank. The transmembrane pressure was normally controlled at 35.15 kg / cm2 with the temperature controlled at 39 ° C-47 ° C. The permeate flow rates varied from 3.3 to 15.3 l / m / hr. The reverse osmosis permeate contained only residual cyclosporin activity and was discarded. EXAMPLE 6 Additional Concentration of Cyclosporin A using Utrafiltration Membrane in Series 1 000 MWCO PLAC of Mil lipore Approximately 20 liters of methanol permeate containing cyclosporin A from the microfiltration of fermentation broth ceramic (Operation CD-273) were placed in a feed tank of a PR ESTA ™ ultrafiltration system from Millipore. A PLAC regenerated cellulose membrane of 0.93 m2 with approximately 1,000 MWCO was used in a tangential flow, plate and frame type module. The current temperature was maintained at 28-30 ° C. The temperature of the stream was maintained at 28-30 ° C using a heat exchanger with cooling water. The inlet and outlet pressures were controlled at approximately 5.62 and 4.78 kg / cm2 respectively and the transmembrane pressure (PTM) was controlled at approximately 3.8 kg / cm2. The permeate flow rate was started at 1 1 .6 L / m2 / hr and finished at 8.4 l / m2 / hr after a concentration increased 5 times (ie, 4 liters of final retentate). The product was retained in the retentate and methanol in the permeate was removed. The normal product yield for this membrane concentration step was approximately 94% with 6% of the product lost in the permeate. The system was then rinsed and cleaned with fresh methanol followed by distilled water to restore the initial flow rate.

Claims (15)

1. REVIVAL DICATIONS 1. A process for recovering a water-insoluble compound from a raw fermentation broth, comprising the steps of: a. concentrating said fermentation broth by tangential filtration through a porous filter membrane compatible with solvent, to produce a permeate traversed said membrane and a retentate comprising said concentrated broth, said water-insoluble compound being retained in such retentate wherein said retentate is it continuously recirculates along a circulation path to form a retentate stream, wherein said raw broth feeds such a retentate stream until all said raw broth is concentrated; b. solubilizing said water-insoluble compound from said retentate by adding a solvent to said concentrated broth to produce a solution of said compound; and c. filtering or diafiltering said solution through said filtration membrane of step (a) to produce a solvent permeate passing through said filtration membrane wherein said solvent permeate comprises said solubilized compound.
2. 2. The process of claim 1, wherein said compound is selected from the group consisting of an immunosuppressant, an acrylic acid antibiotic, an anti-hypercholesterolemic agent, a cyclosporin and its derivatives and intermediates.
3. 3. The process of claim 2, wherein said compound is an immunosuppressant selected from cyclosporin A, cyclosporin B, cyclosporin G, rapamycin, ascomycin and tracolimus.
4. 4. The process of claim 3, wherein said immunosuppressant is cyclosporin A.
5. 5. The process of claim 2, wherein said compound is an antihypercholesterolemic selected from lovastatin, pravastatin, simvastatin and fluvastatin.
6. The process of claim 2, wherein said compound is a macrolide antibiotic selected from erythromycins, A, B, C and D.
7. 7. The process of claim 1, wherein said filtration membrane has a pore size of from about 0.001 μm to about 5.0 μm.
8. The process of claim 1, wherein said filtration membrane has a pore size of about 0.001 μm to about 0.05 μm.
9. The process of claim 1, wherein said filtration membrane is selected from the group consisting of cellulose, polystyrene, polysulfone and polyamide.
10. The process of claim 1, wherein said filtration membrane is ceramic alumina.
11. The process of claim 10, wherein said filtration membrane has a pore size from about 0.05μm to about 5.0μm.
12. 12. The process of claim 1, wherein said solvent of step b is selected from the group consisting of lower alcohol, lower ether, lower ether and lower ketone.
13. 13. The process of claim 12, wherein said solvent is an alcohol selected from methanol, ethanol, propanol and butanol. The process of claim 1, further comprising the step of diafiltering said concentrated broth through said filtration membrane of step (a) before solubilizing said compound in step (b). The process of claim 1, further comprising the step of concentrating said solvent permeate in a reverse osmosis or ultrafiltration membrane.